Developing a Collection of Immobilized Nucleoside Phosphorylases for the Preparation of Nucleoside Analogues: Enzymatic Synthesis of Arabinosyladenine and 2′,3′‐Dideoxyinosine

Abstract

The use of nucleoside phosphorylases (NPs; EC 2.4.2.n) represents a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. We purified four recombinantly expressed nucleoside phosphorylases from the bacterial pathogens Citrobacter koseri, Clostridium perfringens, and Streptococcus pyogenes (CkPNPI, CkPNPII, CpUP, SpUP) and their substrate specificity was investigated towards either natural pyrimidine or purine nucleosides and some analogues, namely, arabinosyladenine (araA) and 2′,3′‐dideoxyinosine (ddI). A 2–3 % activity towards these latter compounds (compared to the natural substrates) was observed. Enzyme activities were compared to the specificities obtained for the enzymes pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP) and purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNPII) previously reported by some of the authors. The enzymes displaying the suitable specificity for the synthesis of araA and ddI were immobilized on aldehyde–agarose. The immobilized preparations were highly stable at alkaline pH and in the presence of methanol or acetonitrile as cosolvent. They were used in the synthesis of araA and ddI by a one‐pot, bienzymatic transglycosylation achieving 74 and 44 % conversion, respectively.

Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine

“…Lastly, in order to obtain a highly active biocatalyst, the enzyme load was increased, obtaining a very good residual activity at the end of the complete immobilization process in presence of 20% of glycerol (56%) ( Table 2, entry 11). A similar behavior was described for the hexameric pyrimidine nucleoside phosphorylase from Clostridium perfringens (CpUP), since uracil was added in order to stabilize this multimeric enzyme during covalent attachment to aldehyde-agarose, but no improvement in the percentage of recovered activity could be achieved [5,8]. On the other hand, the protective effect of glycerol during reduction was also observed in the immobilization of different PNP and PyNP on aldehyde agarose [5].…”

“…The use of substrates or inhibitors [36], as well as polyols [37], has been previously reported for preserving the 3D structure of several enzymes during immobilization, including multimeric nucleoside phosphorylases [5]. The interaction with these additives allows the enzyme to maintain a compact conformation and, thus, reduces the distortion of the 3D structure occurring during covalent immobilization or crosslinking.…”

“…These enzymes are used to catalyze, in presence of inorganic phosphate, the transfer of the sugar moiety from a sugar donor nucleoside to a sugar acceptor base leading to the production of a second nucleoside through a two-step reaction. Moreover, several NPs from different sources have been also conveniently immobilized by following various approaches and obtaining, in most of cases, robust biocatalysts for preparative reactions [5,[8][9][10]. However, the poor ability of NPs to recognize cytosine derivatives as substrates [11] and, often, the need to follow a two-enzyme one-pot scheme (PyNP-PNP or UP-PNP) to synthesize purine nucleosides, represent the main obstacle to their broad application.…”

“…To this aim, the use of nucleoside phosphorylases (NPs) has been extensively described [4][5][6][7]. These enzymes are used to catalyze, in presence of inorganic phosphate, the transfer of the sugar moiety from a sugar donor nucleoside to a sugar acceptor base leading to the production of a second nucleoside through a two-step reaction.…”

Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

“…Immobilisation also allows for a convenient and easier recovery of the biocatalysts and simplifies the design of a reactor. [22] Considering the multimeric nature of the enzyme, [23] and some examples where NPs were successfully immobilised on a hydrophilic support (e.g., epoxide), [24] we chose MagReSyn microspheres as an immobilisation carrier. These microspheres are made of magnetite particles combined with a hydrophilic polymer with epoxide-functional groups, [25] [a] Solubility was measured by UV spectroscopy at the maximum wavelength of each compounds (284-288 nm) in 2 mM potassium phosphate buffer (pH 7.0).…”